LOCUS       sequence-311733-        3008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  12 designed non-target sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3008)
  AUTHORS   Moreb EA, Hutmacher M, Lynch MD
  TITLE     CRISPR-Cas ""Non-Target"" Sites Inhibit On-Target Cutting Rates.
  JOURNAL   CRISPR J. 2020 Dec;3(6):550-561. doi: 10.1089/crispr.2020.0065.
  PUBMED    33346713
REFERENCE   2  (bases 1 to 3008)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1089/crispr.2020"; journalName: "CRISPR J"; date: "2020-12"; 
            volume: "3"; issue: "6"; pages: "550-561"
COMMENT     Sequence Label: pNT-12
FEATURES             Location/Qualifiers
     source          1..3008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      111..142
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        143..245
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             246..1061
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(319..338)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1082..1109
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1121..1708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1201..1220)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1730..1759)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 ggataaggtg ttgattggac gattacagtc aggaaccagc ctcagtgacg aattctctag
       61 atatcgctca atactgacca tttaaatcat acctgacctc catagcagaa agtcaaaagc
      121 ctccgaccgg aggcttttga cttgatcggc acgtaagagg ttccaacttt caccataatg
      181 aaataagatc actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag
      241 ctaaaatgag ccatattcaa cgggaaacgt cttgctcgag gccgcgatta aattccaaca
      301 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
      361 caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
      421 gtagcgttgc caatgatgtt acagatgaga tggtcaggct aaactggctg acggaattta
      481 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
      541 ctgcgatccc agggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
      601 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
      661 gtccttttaa cggcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
      721 gtttggttgg tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
      781 ggaaagaaat gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt
      841 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
      901 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
      961 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     1021 ataaattgca gtttcacttg atgctcgatg agtttttctg agggcccaaa tgtaatcacc
     1081 tggctcacct tcgggtgggc ctttctgcgt tgctggcgtt tttccatagg ctccgccccc
     1141 ctgacgagca tcacaaaaat cgatgctcaa gtcagaggtg gcgaaacccg acaggactat
     1201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1561 gaacagtatt tggtatctgc gctctgctga agccagttac ctcggaaaaa gagttggtag
     1621 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     1681 gattacgcgc agaaaaaaag gatctcaaga agatcctttg attttctacc gaagaaaggc
     1741 ccacccgtga aggtgagcca gtgagttgat tgcagtccag ttacgctgga gtctgaggct
     1801 cgtcctgaat gatatcaagc ttgaattcgt tttctgtggc acgacttctc tggactgttc
     1861 ttatcaactc cgagggtaga ctcaatctga atacttttcg ttactggacc ctggccgtgg
     1921 ggtaaaacgg ttggcgaact acgatacgga agtaacagtc tccctattat tctgattggt
     1981 agcagttaac tatcggtcta accctggccg tgtgggtggg cgtctttgag aactgttgac
     2041 tacacgctta tctgccgaaa tagaatactg agtgccttta taaacggttg actaccctgg
     2101 ccgtgaggta agtagatttc ttcggaggaa taatcgctga gagtcactga aatcaatagt
     2161 atcacacctg tttacgactt tactgaaccc tggccgtgcg ggaaacctgt tatctaccga
     2221 ttcagaagtt cctattgagt tactgggaga ctatcatagt tgccagagtg aatccctata
     2281 ccctggccgt gcgggagaac tttcagccac tatcacggta gaagaatcct tttcggagta
     2341 ttttattggt attcacgcaa ttgccaacag ttaccctggc cgtgtggttg attcggactg
     2401 cgatagtaaa ctgtagataa taagaccgct ctgacaatac ctcaggtttt acgactatcc
     2461 agttaaccct ggccgtgagg ctcagtaaac gaaataatac gataagtagt tcccaatctc
     2521 tggttgtctc ggtagactca gactaattgt tacggacaac cctggccgtg gggtacgaag
     2581 actgattgag gacgcaacta caggttctat tttacttatt acgccagata ggtaaaccag
     2641 aaatcctcgt taccctggcc gtgcggactc tgaaaatccg tgttgaataa aggtgtagta
     2701 gccgttacgc aataccaatc tgaatacact tataataccg tgtcaccctg gccgtgagga
     2761 aacactggat tgacttttca ggaagattcg caactcgtag aagcgtgatt actagttaca
     2821 acgacgaaag cgtatttacc ctggccgtgt ggtaagatag taataaccaa ccctgtttcc
     2881 gctgtagtgt aagtgccttt ctgaagaatt caatcgtaat ctattggcga accctggccg
     2941 tggggagaca ataggatttc gctggtttca ctctcggaat aaagttctca gtagactcaa
     3001 agacgact
//
